1. Field of the Invention
The present invention relates generally to the field of immunotoxins, i.e., toxins conjugated to targeting molecules, and to methods for their preparation and use. The invention particularly concerns novel methods of purifying immunotoxins and determining the most effective species of immunotoxin conjugates for treatment of specific diseases.
2. Description of the Related Art
Ricin A chain (RTA) is the catalytic subunit of the plant toxin ricin and is frequently used for the construction of immunotoxins (ITs) (Lord et al., 1988). To be pharmacologically active, RTA-Its must have a disulfide bond between the antibody and the RTA (Masuho et al., 1982). The chemical linkage of RTA to antibody involves the introduction of activated thiol groups into the molecule of antibody and the reaction of the thiolated antibody with reduced RTA. The free thiol groups of the RTA molecule displace the pyridyl disulfide from the activated thiol groups of the antibody to form disulfide linkages. The resulting IT is then purified to remove the free RTA and antibody by two successive chromatographic steps on Blue-SEPHAROSE SEPHACRYL S-200 (Ghetie et al., 1988a; 1991).
Unfortunately, the IT that results from such methods is not a homogeneous product, but contains mixtures of conjugates comprising several molecular species with one, two or more RTAs linked to each molecule of antibody. These mixtures are not easily separated, particularly during scale-up, because their MWs and electrical charges are similar.
This formation of mixtures of conjugates that contain different proportions of deglycosylated A chain (dgA) or other toxin is the principal problem in the preparation of ITs by chemical methods. The mixture usually comprises a major component containing equimolar amounts of antibody and toxin and several other conjugates with two or more toxin molecules per molecule of antibody. This heterogeneity results from the introduction of various numbers of thiol groups per molecule of antibody. Due to the nature of the chemical reaction between the N-succinimidyl group of the crosslinker and the primary amino groups of the antibody, populations of antibody molecules substituted with none, one, two, three and even more than three thiol groups are obtained.
For example, it was reported that when the crosslinker N-succinimidyl-oxycarbonyl-.alpha.-methyl-(2-pyridyl-dithio)toluene (SMPT) was added to the molecule of antibody (mouse IgG.sub.1) in a 5-fold molar excess, the average number of thiol (MPT groups) introduced was 2 (Ghetie et al., 1991). However, when the IgG.sub.1 -MPT was chromatographed through an activated thiol-AGAROSE column in the inventors' laboratory, 25% of the protein did not bind and after treatment with dithiothreitol, it did not release pyridine-2-thione, demonstrating that 25% of the IgG.sub.1 molecules were not substituted. This indicated that the average number of MPT groups introduced was 2.7 and not 2. Further, when this thiolated antibody was reacted with dgA, the main component (50%) contained one molecule of dgA per molecule of antibody. This suggested that not all MPT groups introduced into the IgG.sub.1 molecule are able to bind dgA and therefore, that the IT may contain some molecules of antibody with unreduced MPT groups.
To decrease the number of MPT groups introduced into the molecule of antibody, a lower molar ratio between SMPT and IgG.sub.1 was used (2-3). In this case, the degree of substitution averaged 1 MPT group per molecule of IgG.sub.1 and a 180 kDa IT was formed after reacting with dgA. However, the yield of IT obtained (10-15%) was considerably lower than the yield obtained when an IgG.sub.1 -MPT with 2 MPTs per molecule of IgG.sub.1 was used (50-60%) (Ghetie et al., 1991). Therefore, merely adjusting the molar ratio between SMPT and IgG.sub.1 is not an efficient way to produce ITs of a particular toxin to antibody molar ratio.
This inability to produce pure antibodies of a particular toxin/antibody ratio has also made it difficult to assess which immunotoxin preparation would be the most beneficial in a pharmacological preparation. Although a comparison of two ITs constructed with either one or two molecules of intact ricin (A+B chains) has been reported (Marsh and Neville, 1986), this comparison has not been done with the ricin A chain conjugated to an antibody. Therefore, there exists an immediate need for a procedure for large scale, efficient preparation of immunotoxins which can be separated according to the toxin/antibody molar ratio.